| Background and objective:The present study showed that cerebrovascular disease is one of the important diseases caused to human death. Intracerebral hemorrhage(ICH), as higher mortality and morbidity, is accounted for about 15-20% of stroke. Epidemiological data showed that the new cerebrovascuar disease patients were about 2 million people each year. cerebrovascuar disease cause about 1.5 million patients to deaths. About 6 million survivors were mostly suffered from various neurologic impairment such as hemiplegia, aphasia and dysphagia, and were oppressively burdened by the society and family. Past research indicate that the main cause of neruons injury after ICH were brain ischemia and edema mostly caused by microcirculation damage in regional around the hematoma and the hematoma pressure. But present study found, in addition to the hematoma compression, that release of active substances after intracerebral hemorrhage were involved in the process of brain injury. Minimally invasive surgery (MIS) is a kind of removal technology of intracranial hematoma, which characterized by combination of drill cranial, piercing and fixed. MIS combined with fluid mechanics principle and enzyme technology can effectively remove the hematoma.MIS currently has been widely used in clinical by virtue of alleviate trauma, quickly reduce intracranial pressure and rapidly recover neural function. But MIS can not inhibit blood breakdown products damage nerve cells of hematoma peripheral. A number of studies have confirmed that therapeutic hypothermia is one of the most promising neural protection method, its efficacy has been widely recognized. Hypothermia treatment were able to alleviate neuronal injury after ICH through multiple approaches. In order to reduce the body temperature, Some doctors have used to hypothermia treatment through artificial hibernation therapy. However, higher risk of artificial hibernation therapy, such as inhibiting respiratory, limit its clinical application for patients with ICH. At present, methods of hypothermia treat ICH were mostly used to ice cap decrease the temperature of the extracranial. It is difficult to directly intervene in the temperature of the intracranial. Protection mechanism of hypothermia on brain injury after ICH is not also fully clear. Hence, The purpose of this study lies in:to explore the protection mechanism through effect of hypothermia on neuronal Ca2+ channels and apoptosis after bleeding, to proved the protection mechanism of hypothermia on brain injury after ICH through the establishment of animal model, to observe clinical effect of minimally invasive surgery joint local cooling lavage on treatment of ICH.Method:1. Dissociated cultures of hippocampal neurons were prepared from postnatal 0-1 day mice. Glial growth was suppressed by addition of 5-fluoro-2-deoxyuridine and uridine. Mature neurons used for experiments were divide into three groups:Normal control group,37℃ normal temperature group,33℃ hypothermia groups and 25℃ low temperature group. In addition to the control group, cultured neurons of three groups induced injury with whole blood of maternal rats, after 1h, we detect LDH level expressed neurons by ELISE method, detect intracellular calcium concentration by calcium laser confocal imaging and observe counting of neuron apoptosis by laser confocal microscope.2. ICH model was established by type IV collagenase caudatum infusion. The rats were treated with MIS 6 h after injection, then were lavaged by normothermic (37℃) and hypothermic (33℃) normal saline (NL) in brain separately, the rats were sacrificed 72h after ICH induction, brian water content was detected, we measure content of LDH, SOD, IL-1β and IL-10 around perihematoma by Enzyme-linked immunosorbent assay (ELISA). we detect to express of TUNEL by immunofluorescence double staining and observe counts of TUNEL-positive cells by laser confocal microscopy, we also observe hippocampal neuron ultrastructure under an elextron microscope.3.50 cases patients wiht ICH were adopted into our experiments, were divided into MIS group (20 cases) and MIS+LCL group (30 cases). The patients in MIS group were treated by simple minimally invasive surgery, these in MIS+LCL group were treated local cooling lavage with 33℃ saline after treatment of minimally invasive surgery. all adopted patients were measured by means of NIH Stroke Scale(NIHSS) in respectively before and after treatment 1 d,7 d and 14 d, evaluated recent curative effect by means of glasgow outcome scale (GOS) after a month,and estimated the long-term prognosis through activities of daily living(ADL) after 6 months.Result:1. The results shows that the LDH levels of hippocampal neurons injured by whole blood is obviously increased. The release of LDH in cultured hippocampal neurons in the 33℃ hypothermia group was numerically less than those in 37℃ normothermic group (P＜0.05). In control groups, Neurons apoptosis in control groups were not found in the field of vision. In the 33℃ hypothermia group, we found injured neurons, which were Neuronal shape pyknosis, uneven chromatin and obscure internal structure. The proportion of injured neurons in all neurons were 43±11.1% in the 33 ℃ hypothermia group. In the 37℃ normothermic group, the proportion of injured neurons in all neurons were 60±10.5%.There were obvious difference between above two groups. The proportion of injured neurons in 25℃ low temperature (83+13.5%) was highest, the ca2+ concentration of hippocampal neurons injured by whole blood is obviously increased. The intracellular ca2+ concentration in the 33℃ hypothermia group was clearly less than those in 37℃ normothermic group(P＜0.05).2. Experiment of ICH rats model induced by collagenase:(1)Hematoma volume measurement:The brain of the rats in ICH, MIS and NL groups had a cavity within the center of the lesion. The hematoma volume was respectively 28.3 ± 2.8 ml in ICH group (n=5),10.7 ± 3.2 ml in MIS group and 8.4± 2.5ml in MIS+LCL group (n=5). The hematoma volume was significantly reduced by MIS (P＜0.05, Fig.1). But the combined treatment with MIS and LCL exhibited no less hematoma volume in cerebral of the rats in ICH.(2) The mNSS scores respectively were 12.8 ± 1.3 in the ICH group,8.8 ± 1.0 in the MIS group,8.6 ± 1.1 in the MIS+NL group and 6.1 ± 0.9 in the MIS+LCL group (n=5). MIS may alleviated neurological deficit of the rats after 3 days, but NL did not further improve neurological function. Whereas mNSS scores in MIS+LCL group displayed less than that of MIS group。(3) HE staining revealed a high density of inflammatory cells within and around the hemorrhagic lesion 3 days after ICH induction. The number of inflammatory cells was similar in both the control group (108.0 ± 41.5 cells/mm2, n=4) and SO group (119.6 ± 53.5 cells/mm2, n=5). MIS reduced the number of inflammatory cells in the perihematomal area. The numbers of inflammatory cells infiltration in MIS group (319.1 ± 60.6 cells/mm2, n=5) is visibly reduce compared with those in ICH group (429.6 ± 63.2 cells/mm2, n=5). Local infiltration by inflammatory cells at the periphery of the hematoma in LCL group (249.8 ± 58.5 cells/mm2, n=4) is remarkable decrease compare with ICH and MIS groups. In addition, Around the hematoma organization structure in cerebral of the rats in ICH Suffered serious damage. Thus, this kind of damage were prevented by MIS. Furthermore, The treatment of LCL reduce greatly injury of around the hematoma compare with only treatment of MIS.(4) Expressions of SOD, LDH, IL-1β and IL-10:At 3d after ICH, The expressions levels of SOD in rat’s brain tissues in the MIS group were not obvious difference compared with in ICH group. We also observed that SOD immunoreactivity in MIS+LCL group was stronger than one in MIS group. The expressions levels of LDH in rat’s brain tissues in the MIS group were numerically less than those in ICH group (P<0.05). Levels of LDH in MIS+LCL group were least (P<0.05). In MIS group, the expressions of IL-10 in rat brain tissues were also numerically more than those in the ICH group (P<0.05). The expressions levels of IL-10 in rat’s brain tissues in the MIS+LCL group were not obvious difference compared with in MIS group. These data indicate to MIS alleviate inflammatory response by enhancing expression of IL-10. But LCL may not effect on expressions levels of IL-10. On the contrary, the expressions levels of IL-1β in rat’s brain tissues in the MIS group were significantly decrease compare with those in ICH group (P< 0.05). In MIS+LCL group, the expressions of IL-1β were also numerically less than those in the MIS group (P<0.05). Thus, MIS treatment reduced inflammatory response by inhibiting expressions levels of IL-1β. on the basis of MIS, Moreover, MIS joint LCL treatment further alleviate inflammatory response in hemorrhagic lesion of the rat’s brain in ICH. But NL treatment have not changes both expressions levels of IL-10 and IL-1β.(5) Neuronal apoptosis detection:the positive cells of TUNEL of around the hemorrhagic lesion of the rat’s brain were most in ICH group. The numbers of TUNEL positive cells in MIS group were obviously less than those in ICH group (n=3, P<0.05), But MIS joint NL treatment not reduce neuronal apoptosis. Moreover, The expression of TUNEL positive cells of the rat’s brain in MIS+LCL group were decreased greatly compared with MIS and MIS+NL groups (P＜0.05). These results imply that LCL joint MIS can inhibited further neuronal apoptosis of the rats in ICH.3. Clinical effcet observation NIHSS score of the patients in MIS LCL group compare with MIS group showed a decreasing trend Id,7d after treatment, but without statistical significance.14 days after treatment, NIHSS score of the patients in the MIS+LCL group were significantly less than one in MIS group (P＜0.05). From the recent clinical curative effect respects, total effective rate of MIS+LCL group (86.7%) was obviously higher than the MIS group (75.0%), the difference was statistically significant (P＜0.05). ADL grading comparison of two groups after 6 months:Optimal,good and middle residual rate in the MIS+LCL group (80.0%) is significantly higher than in the MIS group (70.0%). On the contrary, the heavy disability in the MIS+LCL group (20.0%) was lower than that in group MIS (30.0%), the difference was statistically significant (P< 0.05).Conclusion:1. By culturing mouse nerve cells in vitro, confirmed that hypothcrmia can alleviate release of LDH and reduce neuron apoptosis. And proves that hypothermia were able to decreaseintracellular calcium overload through the application of calcium imaging experiments.2. This study demonstrated that the strategy of using MIS joint LCL may achieve the hematoma volume after ICH, reduce tissue structure damage around hematoma lesion. MIS joint LCL may enhance neuroprotection against neurons apoptosis after ICH by inhibite Oxidative stress and inflammation, and enhance antioxidant and anti-inflammatory.3. MIS joint LCL treatment can alleviate with nerve function defect, improve the recent and long-term curative effect, enhance the quality of survival,and reduce morbidity of patients with ICH. |
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